LOCUS       sequence-455404-        6551 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express node 1438 retron with split retron ncRNA and gRNA to edit 
            (10bp barcode) EMX1 gene in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6551)
  AUTHORS   Khan AG, Rojas-Montero M, Gonzalez-Delgado A, Lopez SC, Fang RF, 
            Crawford KD, Shipman SL
  TITLE     An experimental census of retrons for DNA production and genome 
            editing.
  JOURNAL   Nat Biotechnol. 2024 Sep 17. doi: 10.1038/s41587-024-02384-z.
  PUBMED    39289529
REFERENCE   2  (bases 1 to 6551)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2024 Sep 17. doi: 10.1038/s41587-024-02384-z."
COMMENT     Sequence Label: pAGK60
FEATURES             Location/Qualifiers
     source          1..6551
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        368..645
                     /label=chicken beta-actin promoter
     intron          646..1662
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1586..1608
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1670..1689
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2768..2788
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2816..3040
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3075..3315
                     /label=U6 promoter
                     /note="Truncated RNA polymerase III promoter for human U6 
                     snRNA used in pSIREN vectors such as 
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site 
                     is 8bp downstream of this feature."
     primer_bind     3075..3095
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3246..3265
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3682..3896
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     3817..3836
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(4036..4052)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4036..4052)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4049..4071)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4060..4076
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4084..4090,4091..4108,4109..4114))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4129..4150
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4209..4404
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4255..4390
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4410..4544
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4447..4466)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4501..4520
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4612..4629)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4783..5371)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4863..4882)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5542..6333,6334..6402))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6165..6184
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6403..6507)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        join(6538..6551,1..366)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
       61 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      361 accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca
      421 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
      481 gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg
      541 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
      601 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc
      661 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
      721 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
      781 cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc
      841 cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg
      901 gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg
      961 gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg
     1021 tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga
     1081 gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc tccccgagtt
     1141 gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc
     1201 gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc
     1261 ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg
     1321 gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg
     1381 tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc
     1441 ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg
     1501 ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc
     1561 cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga
     1621 gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg
     1681 gttattgtgc tgtctcatca ttttggcaaa gaattgagct cgatatcggt acgccaccat
     1741 gaagaacaat gacatcaact catggatgga gtttttcctg gaccgtggag tgtcctctga
     1801 ccaaatcaac tattacatcc cgtacatcgc caagctcctg aagactaaca gtcctattat
     1861 cttcgagaaa cagcacctgg cccatcttat cggtattgac gaatctatcc tggataagat
     1921 tatctctagc cccaatcatt tttatcgtga gtttttcatt cctaaaaagc gcggaggcct
     1981 caggaagatc acttgtccgt accctagtct cctgatgtgt caaaagtgga tctataagaa
     2041 catcctcctg aacgacaccc cacacgtctc ctgtcatgga tttaccccga gcaagtctat
     2101 tatcaccaac gcttcccttc accttaagca gaagtgtttg ctgaagatgg atgtgaagga
     2161 ctttttcccg tccattccaa tcaactgggt gattaactat tttaaaggtc tgggatacag
     2221 tgataacctg agcttctata tgagctccct gtgctgtcac gataaaaagc ttgctcaggg
     2281 cgcttccaca agcccgtacc tttccaatct tctgttgaag ggcctcgata accggctgaa
     2341 taacctgtcc cgcaagtacc gcctgaccta tagtcggtac gcggatgaca tgcatttctc
     2401 aggtgattac atcccctctg acctggtggg cgttattaca tcaattatca ataactacgg
     2461 gctttccgtt aatgagaaga aaacagctct gtatatcaag tcaaataagc gcatcattac
     2521 aggaatctca gtggccggtg atgagctgtc cctgccccgc gactacaaga gaaagctgaa
     2581 aaacgagctg catttcattg agaagttcgg ttacatctct cacattaccc agaagaaaat
     2641 caaggaccca aactacctgt attccattct gggtcgcgtg aacttctggc tgcagatcga
     2701 gccagacaac gatgctgcca tcaaggggag ggactatctg atgcgtgtcc ttaagaacaa
     2761 tctgccacca aagaagaaaa gaaaggtctg atcccggtta gtaatgagtt taaacctgtg
     2821 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     2881 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     2941 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     3001 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg agatctgcga aatcggatcc
     3061 agctgaatct aagtgagggc ctatttccca tgattccttc atatttgcat atacgataca
     3121 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
     3181 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     3241 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     3301 tcttgtggaa aggacgaaag gaatggctta cagaagctag tatgttagag tggagtcgct
     3361 ctcatgataa ggtctgggtt aacccagttc atggaaatta ctgtaaccag taatttccat
     3421 gatgtggata ataaaatcac aaacggcaga agctggagga ggaagggcct gagtccgagc
     3481 agaagaactg ggggataaaa gttctcccat cacatcaacc ggtggcgcat tgccacgaag
     3541 caggccaatg gggaggacat cgatgtcaga ttttattatc tggattggac cagcaaattt
     3601 ttgcgttttc gcaaaatttt gccgtcccaa tccacaggct aaagtattga aggtatcttc
     3661 tgtaagccat ttcttttttt tgaacgctga cgtcatcaac ccgctccaag gaatcgcggg
     3721 cccagtgtca ctaggcggga acacccagcg cgcgtgcgcc ctggcaggaa gatggctgtg
     3781 agggacaggg gagtggcgcc ctgcaatatt tgcatgtcgc tatgtgttct gggaaatcac
     3841 cataaacgtg aaatgtcttt ggatttggga atcttataag ttctgtatga gaccacgaaa
     3901 gagtccgagc agaagaagaa gtttcagagc tatgctggaa acagcatagc aagttgaaat
     3961 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttttttttag
     4021 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4081 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4141 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4201 gcggatccgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     4261 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     4321 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     4381 tttggaggcc taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca
     4441 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4501 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat
     4561 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4621 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4681 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4741 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4801 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4861 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4921 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4981 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5041 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5101 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5161 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5221 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5281 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5341 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5401 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5461 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5521 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5581 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5641 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5701 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5761 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5821 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5881 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5941 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6001 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6061 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6121 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6181 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6241 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6301 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6361 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6421 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6481 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgggtcgac
     6541 attgattatt g
//
